Nucleic acid chemistry
part 1 : Some aspects of chemical and
enzymatical modification
of nucleic acids
Chelating agents :
EDTA
ethylene diamine tetraacetic acid
EGTA
ethylene glycol tetraacetic acid
(ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid)
chelating agent with stronger affinity to
calcium than magnesium ions than EDTA
CDTA
trans-1,2-diaminecyclohexane-tetracetic acid
(weaker chelator, used to remove Ca2+ from cell walls)
NTA
nitrilotriacetic acid
(chelating Ca2+, Fe2+, Cu2+, ...)
In contrast to EDTA, it
is easily biodegradable.
Carcinogenic in different animal model
systems, hence possibly carcinogenic to humans.
A modified NTA is used to immobilize nickel to a solid
support, and used in the His-tag method to purify
recombinant proteins, with 6-His at the C-terminus.
Structure of the complex of NTA3- and Ca2+
Intercalation
Ethidium bromide :
UV absorption curve of double-stranded DNA
Spectra of the
four individual
nucleotides A, C, G, T
1 A260 of DNA = 50 mg/ml
1 A260 of RNA = 40 mg/ml
Extinction coefficient, single-stranded DNA
e260 = 6100 mol-1 x L-1
relative contributions :
A > G
1.52
1.21
> T > C
0.84
0.705
The shape of the absorbance curve of Adenine resembles best the one of large DNA (is somewhat
thinner) ; the curves of Guanine, Thymine and Cytosine are more irregular, making an overall
DNA curve a bit broader, but remaining nicely symmetrically shaped with a maximum close to 260
nm. There is a dip between 220 and 230 nm. Above 320 nm there is no more absorbance, and
below 220nm (in the far UV) the solution becomes opaque.
At 260 nm, the UV absorption (OD, optical density) of single stranded DNA is
30 to 40% higher than when base-paired into a double helix.
At 280, DNA absorbs about 50% less than at 260 nm, hence DNA has an 260/280 absorbance
ratio of 2. (Above 1.7 a sample is considered being pure.)
Proteins have a maximum at 280 nm (especially due to the aromatic side- chains).
Contamination with protein will lower the 260/280 ratio below 2, although this usually
requires a high level of contaminating protein. In contract, contamination of protein samples
with (small amounts of) DNA (or RNA) are readily detected.
Other UV-absorbing substances, e.g. phenol may disturb concentration measurements
substantially.
With shorter strands, the A260 value lowers :
Nucleic Acids
A260
Double-stranded DNA
50
Single-stranded DNA or RNA (>100 nucleotides)
40
Single-stranded oligos (60–100 nucleotides)
33
Single-stranded oligos (<40 nucleotides)
25
(e.c.L)
Protein contamination and the 260:280 ratio
The ratio of absorptions at 260 nm versus 280 nm is commonly used to assess DNA
contamination of protein solutions, since proteins (in particular, the aromatic amino acids) absorb
light at 280 nm. The reverse, however, is not true : it takes a relatively large amount of protein
contamination to significantly affect the 260:280 ratio in a nucleic acid solution.
260:280 ratio has high sensitivity for nucleic acid contamination in protein:
% protein
% nucleic acid
260:280 ratio
100 0
0
0.57
95
5
1.06
90
10
1.32
70
30
1.73
260:280 ratio lacks sensitivity for protein contamination in nucleic acids (table shown for
RNA, 100% DNA is approximately 1.8):
% nucleic acid
% protein
260:280 ratio
100
0
2.00
95
5
1.99
90
10
1.98
70
30
1.94
This difference is due to the much higher extinction coefficient nucleic acids have at 260 nm
and 280 nm, compared to that of proteins. Because of this, even for relatively high
concentrations of protein, the protein contributes relatively little to the 260 and 280
absorbance. While the protein contamination cannot be reliably assessed with a 260:280
ratio, this also means that it contributes little error to DNA quantity estimation.
Other common contaminants
Contamination by phenol, which is commonly used in nucleic acid purification, can
significantly throw off quantification estimates.
Phenol absorbs with a peak at 270 nm and a A260/280 of 1.2.
Nucleic acid preparations uncontaminated by phenol should have a A260/280 of around 2.
Contamination by phenol can significantly contribute to overestimation of DNA
concentration.
nucleotide bases
5-bromo uracil
5-methyl cytosine
5-hydroxymethyl cytosine
5-hydroxymethyl thymine
5-hydroxymethyl thymine, glucosylated (1 or 2)
etc.
2’-deoxynucleoside
ribonucleoside
intramolecular base pairing
stem-loop structure, bulge,
interior loop
(always in 5’-3’ orientation)
3’-5’ cyclic adenosine phosphate
Western alphabet :
ABCDEFGHIJKLMNOPQRSTUVWXYZ
The names "nucleoside" for pentose + base,
and "nucleotide" for phosphate + pentose + base:
were introduced by P.A.Levene & W.A.Jacobs in 1909.
Nomenclature of nucleosides
(deoxy)adenosine
(deoxy)guanosine
(deoxy)cytidine
(deoxy)thymidine
(deoxy)uridine
nucleotides (nucleoside phosphates)
(deoxy)adenosine phosphate or (deoxy)adenylate
(deoxy)guanosine phosphate or (deoxy)guanylate
(deoxy)cytidine phosphate or (deoxy)cytidylate
(deoxy)thymidine phosphate or (deoxy)thymidylate
(deoxy)uridine phosphate or (deoxy)uridylate
One-letter symbols :
A, G, C, T (originally U for uridine in RNA, now also T accepted as symbol) (or dA, dG, dC, dT)
for RNA or DNA with inosine as nucleoside (hypoxanthine as base) : symbol I.
The symbol for any nucleoside is N
(X for an undefined (or unknown) nucleoside)
for other positions with multiple choices : following standard symbols :
R : A or G (puRine nucleoside)
Y : T or C (pYrimidine nucleoside)
S : G or C ('Strong' basepair)
W : A or T ('Weak' basepair)
M : A or C (aMino functional group)
K : T or G (Keto functional group)
B : C or T or G ( = no A)
D : A or T or G ( = no C)
H : A or C or T ( = no G)
V : A or C or G ( = no T)
N
N
aS-thiophosphate analog
N
2', 3' dideoxy analog
3'-deoxyribonucleoside triphosphate = cordicepin triphosphate is another analog.
base pairing
At the isotopic level
Alternative isotopes (underlined=stable isotope) or radioactive labeling (b-radiation)
31P
12C
14N
H
32S
127I
<=> 32P, 33P
<=> 13C, 14C
<=> 15N
<=> T (3H)
<=> 35S
<=> + 36 other isotopes ; (127I is the only stable iodine isotope)
135I
<=>
has a half-live less than 7 hrs, too short for use in biology
129I has a half-live of 15.7 million years ; all others : half-lives less than 60 days)
125I, 131I. (Also 123I and 124I can be used but are not b- emitters ; also shorter half-lives)
Radioactive isotopes often used in molecular biology ;
b--radiation
32P
33P
35S
3H
14C
Other decay
125I
half-life
14.3 days
25 days
87.4 days
12.3 years
5,730 years
max energy
1.710 MeV
0.249 MeV
0.1673 MeV
0.156 MeV
0.018 MeV
average energy
0.70 MeV
0.0769 MeV
0.0492 MeV
0.050 MeV
0.0055 MeV
59.4 days (electron capture)
131I => still occasionally used : half-life 8 days (b- + g emitter)
Chemical changes at the atomic level :
P=O => P=S
(remember : tautomerism !!!)
P-O- => P-S=> S32 may be replaced by the radioactive S35 (b-emitter)
a-S-thiophosphate analog
=> effects on enzymatic activities, such as exonucleases , polymerases,
T4-kinase, etc...
Iodine can replace a H-atom at position C-5 of cytosine
S35 labeling can also be used in proteins. Iodine-labeling is possible as e.g. antibody
conjugates.
Chemical hydrolysis
DNA - is relatively stable in alkali
(some cleavage of A and C rings occurs upon
prolonged incubation in 1M NaOH at 90°C)
(primary structure)
- is acid labile : loss of the base (cleavage of the glycosidic bond)
- DNA > labile than RNA
- purines are lost more easily than pyrimidines
- in formic acid + diphenylamine : depurination reaction (named Burton degradation)
=> leading to oligopyrimidines (also named apurinic acid)
- methylation at G (and much less at A) with dimethylsulfate, followed by treatment
with piperidine also leads to strand cleavage at purine nucleotides
- in hydrazine (hydrazinolysis) : formation of oligopurines
RNA - is hydrolyzed in alkali to mononucleotides, via a 2’-3’ cyclic phosphate intermediate
- most typically, the 2’-3’ ends are sensitive to cleavage by periodate (which targets
cis-diols) and may subsequently undergo a b-elimination (leading to a 3’ phosphate end)
or be derivatized by an alkylamine (and sodium borohydride ; NaBH4).
Formation of apurinic sites in DNA.
In the presence of acid, the
N-glycosyl bond of deoxyadenosine
(or deoxyguanosine) is hydrolyzed,
leading to the formation of the
apurinic site and adenine (or guanine).
Reaction of thymidine
with hydrazine
(similarly with cytidine)
acid : pH 3-4 : loss of purine from the glycosidic bonds
=> then with an amine : cleavage of the apurinic sites
(originally diphenylamine was used by Burton in the so-called Burton degradation)
(other amines may be used, e.g. piperidine (Maxam & Gilbert), or the use of NaOH)
at more highly acidic pH : total hydrolysis of DNA
in principle, DNA is stable in alkali (though obviously denatures to single
strands !) but heating in 1M NaOH leads to partial (random) fragmentation
at A's (and to an even lesser extent at C's (10% relative to A))
Formation of apurinic sites in DNA after alkylation of
deoxyadenosine and of deoxyguanosine by dimethylsulfate.
strand cleavage by heating in 1M piperidine
see Chemical Degradation Sequencing
according to A.Maxam & W.Gilbert
Alkaline hydrolysis of RNA
N
RNA
RNA
N
RNA
N
and
RNA
+
(2'-phosphate)
(3'-phosphate)
RNA
Oxidation of the 3’ terminal nucleoside of RNA (cis diol) with sodium periodate
and subsequent derivatization with an alkylamine. (R = CH3(CH2)n- or NH2(CH2)n-)
N
N
or : b-elimination, leaving a 3'-phosphate
N
The amino groups of the bases are targets for deamination or modification to Schiff bases
Deamination of adenine-, cytosine- and guanine-containing nucleosides (r = ribose or deoxyribose)
Reaction of aldehydes with cytidine, adenosine and guanosine to form Schiff base adducts.
(r = ribose or deoxyribose ; R = H or CH3(CH2)n- )
De-amination : at NH2 positions
- with NaNO2 at around pH 4.5 (via diazonium intermediate derivatives).
- rate : ratios of 1:2:6 for G:A:C
A => I
(mutagenic)
base-pairing to C at replication
C => U
(
“
)
base-pairing to A
G => X
(inactivating)
I and U mimic G and T, respectively.
Reaction with hydroxylamine (NH2OH)
- target position C4 in cytosine : results in hydroxylation of the amino group (and
the C=N double bond) under mild conditions (leads to pairing to A) ;
- results in deamination to U under more vigorous conditions.
- in vitro treatment of DNA => mutagenesis. In both cases leading to CG => TA
Reaction with Na-bisulfite (NaHSO3)
- deamination of C (to U) is thousandfold faster than of purines.
=> same transition point mutations as obtained with hydroxylamine.
- is used in vivo and in vitro.
- with R-NH2 and R(NH2)2
=> 4-alkyl-cytosine and 4-(aminoalkyl)-cytosine
=> possibility of attachment of reporter molecule
Sodium bisulfite-catalyzed deamination of cytidine to uridine in aqueous solution,
and transamination of cytidine to 4-alkylcytidine or 4-amino-alkylcytidine
(r = ribose or deoxyribose ; R = CH3(CH2)n- or NH2(CH2)n-).
(not with 5-methyl-C or 5-hydroxymethyl C)
Alkylating agents :
e.g. (m)ethylmethanesulfonates, (m)ethyl N-nitrosoureas
-
nucleophilic centres in DNA molecules are the N and O atoms in the bases
-
most electron-dense (and hence nucleophilic) : N-7 in G (see above DMS methylation)
other sites: O6 and N-3 in G (less than N-7)
N-7, N-1, N-3 in A
O4 in T
-
in (after) alkali : O6 is the preferential target : O6-alkylation
-
also : alkylation of the phosphate moiety (acidic target!) => phosphotriester
=> subsequently : in strong alkali : either loss of alkyl group or strand cleavage
(in strong acidic conditions : regeneration of the phosphodiester)
Two kinds of reagents:
-
monofunctional : e.g. ethylmethanesulfonate :
=> puts a methyl group on G (at N7 and O6)
=> causing faulty pairing with T
-
bifunctional : e.g. nitrosoguanidine, mitomycin, nitrogen mustard
=> cross-links the DNA strand : faulty region excised by DNase
=> both point mutations and deletions
nucleophilic centres
7
1
6
7
3
3
4
Alkylation of deoxyguanosine at basic pH to form O6-methyldeoxyguanosine.
Alkylation of the phosphodiester internucleotide bond by ethylnitrosourea.
Reaction of guanosine with glyoxal
(r = ribose or deoxyribose)
Reactions with aldehydes, dialdehydes :
- aldehyde : formaldehyde : form Schiff’s bases (or alike) (see also above)
=> derivatives at exocyclic NH2 groups (C, A, G)
=> changes hydrogen bonding abilities of the bases
=> also cross-linking between bases in ds-DNA
- dialdehyde: glyoxal, kethoxal : selective reaction at G
=> formation of an adduct, unable to base-pair (between C and G)
Introduction of modifications by filling-in or copying reactions with DNA or RNA
polymerases and modified dNTP’s or rNTP’s (or ddNTP to add a single nucleotide)
to introduce modified nucleotides.
See also in other parts
Filling-in of a recessed 3’ OH
by polymerase.
Nucleophilic attack onto the a-phosphate.
Release of pyrophosphate.
Remember : antiparallel orientation of the
complementary strands !
Enzymatical tagging with biotin or
digoxigenin by polymerase reactions.
(see also in other parts)
Requirements:
characteristics of the polymerase
exonucleolytic activities ? (5'=>3' , 3'=>5')
processivity ? (cfr. later : "exonucleases")
substrates? : standard dNTP
? rNTP ? (Mn2+ dependent?)
? thiophosphate variants ?
? biotinylated variants ?
? digoxigenin-modified variants ?
? fluorescent modifications ?
? etc...
What is processivity ? : the ability of an enzyme to repetitively continue its
catalytic function without dissociating from its substrate.
with DNA polymerases : the average number of nucleotides added by a
DNA polymerase enzyme per association/dissociation with the template.
DNA polymerases involved in DNA replication tend to be highly processive,
while those involved in DNA repair tend to have low processivity.
In vitro, with low processivity enzymes, more uniform elongation of the DNA
mixture in the sample will be obtained. With high processivity, there will be more
versatility in chain lengths.
Alkaline phosphatases : dephosphorylation (phosphomonoesterases)
- remove phosphate from 5’-, 3’- and 2’-ends of nucleic acid and other
phosphomonoester compounds (including proteins, BCIP, etc.)
- Zn(II) metalloenzymes, which hydrolyze the monoester via a phosphorylated
serine-intermediate
- BAP, CIAP, SAP : stability versus lability
BAP : extremely stable, difficult to inactivate ; E. coli enzyme
(removed by SDS-phenol after treatment at >60°C)
CIAP : calf intestinal enzyme, easier to inactivate
(although still extraction needed to remove it completely)
SAP : from Pandalis borealis (an arctic organism)
completely inactivated by heating at 65°C
- inhibition by inorganic phosphate and chelators of metal ions (EDTA, EGTA)
Some other specific enzymatic modifications :
(not exam material)
DNA ligases : reaction mechanism
or
DNA ligation mechanism
R = ribose
A = adenine
N+ = nicotinamide ; NMN = nicotinamide mononucleotide
E = enzyme (ligase)
Pyrophosphatase : e.g. tobacco acid pyrophosphatase (TAP)
=> cleaves phosphoanhydric bonds e.g. in dNTP, rNTP, CAP structures, …
CAP-structure of eukaryotic mRNA
T4 polynucleotide kinase : 5'-phosphorylation
- transfers the g-phosphate of ATP onto the 5’ end (onto a 5’-OH, or by
an exchange reaction)
- applications : - enables ligations (with e.g. synthetic fragments)
- 5’-terminal labelling / tagging
Terminal deoxynucleotidyl transferase (TdT)
- polymerisation without copying
- monomeric enzyme that adds 5'-mononucleotides to a 3'-OH end of DNA
- may be ss or ds ; minimal starter (p)N-N-NOH
- substrates dNTP, rNTP, ddNTP, …
- a single (d)NTP substrate will yield a (3’-)homopolymic tail
- ddNTP as substrate makes a single addition, also cordicepin triphosphate
(= 3'-deoxy-adenosinetrifosfaat) or rNTP followed by alkali
(!! in which case there is an extra phosphate !!)
Exonucleases :
What is processivity ? : the ability of an enzyme to repetitively continue its
catalytic function without dissociating from its substrate.
(see above, DNA polymerases )
With exonucleases : low or no processivity => more uniform stepwise degradation of the substrate
high processivity
=> more heterogenous mixture of sizes
- exonuclease III (of E.coli)
- active in the excision-repair pathway
- 31 KDa monomer globular protein (gene xth) ; requires Mg2+
- substrate is only ds DNA
active on 5’ extensions of ds DNA
active on blunt ends of ds DNA
also extending nicks to gaps
not active on single-stranded DNA but may be active on
3’ extensions not longer than 3 nucleotides
or 4 nucleotides if the terminal one is a C
- removes stepwise pN from the 3’-OH ends
(or 3'-phosphate ends , since it has some intrinsic 3’-phosphatase activity)
- displays some sequence dependence : C > A = T > G
- end-product = two half-strands (until they fall apart)
- not processive or processive, depending on the reaction conditions
- very efficiently controllable in time or by temperature
=> progressive deletions (under conditions of low processivity)
- resistant against thiophosphate linkages (e.g. fill-in one end with a a-S-dNTP)
- other activities : AP-endonuclease (at apurinic and apyrimidinic sites)
RNaseH
- exonuclease VII
(of E.coli)
- heterodimer proteïn ; 2 genes : xseA and xseB
- active on ssDNA from both (3' + 5') ends
- shortens ss ends of ds fragments but not useful to make blunt ends
(involved in repair mechanisms and homolous recombination)
- not active onto internal loops (hence clearly an exonuclease)
- not sensitive to EDTA (no divalent metal ion required)
- processive
- under limiting conditions :
=> rather long oligonucleotides (> 100 nt) are produced
initially, then further hydrolysed to sizes of 2-12,
but no mononucleotides !
- there may be a phosphate group at either 5'- or 3'-end
- applications (in vitro):
- removal of ss regions
- rapid destruction of excess primers following PCR
- (earlier : study of exon – intron structures)
- bacteriophage l exonuclease
- most active on ds DNA with a 5' phosphate ; (50-250 times better than on ss DNA)
- preferred substrate is blunt-ended, 5'-phosphorylated (but the reaction
mechanism (in initiation) seems to be quite complex)
- removes pN from the 5' end (if 5'-phosphate, greatly reduced rate at 5'-OH ends)
- highly processive
- requires Mg2+
- not active on a nick or a gap
- phage T7 exonuclease (phage T7 gene 6 product, discovered 10 years after l exonuclease)
- removal of 5'-mononucleotides from the 5' end
- active on ds DNA, regardless of 5'-phosphate or 5'-OH. (Barely active on ss DNA)
- also initiates at nicks and gaps in a duplex DNA
- seems also to degrade DNA and RNA of DNA/RNA hybrids but is not active
on either ds or ss RNA.
- low or no processivity
=> more uniform degradation than with l exonuclease
- M2+ absolutely required (5 mM Mg2+ or 1 mM Mn2+) also a sulfhydryl agent (DTT)
- phage T5 exonuclease (phage T5 gene D15 gene productfound 10 years after l exonuclease)
- initiates nucleotide removal from 5' ends, gaps and nicks, of linear and circular
ds DNA.
- degradation in 5' to 3' direction
- supercoiled DNA (cccDNA) is not degraded.
- application : degradation of linear ssDNA, dsDNA or nicked plasmid DNA
while preserving supercoiled plasmid DNA.
- ss DNA endonuclease activity is suppressed by lowering Mg2+ concentration to
less than 1mM
Many other exonucleases have been described, some of which have also been further developed (and
commercialized) for DNA manipulations. E.g. thermoresistant enzymes.
Known and used since the 1960-70's, as they are active on DNA and RNA, are the following :
- snake venom phophodiesterase : cleaves off pN
- exonucleolytically from a 3'-OH of DNA or RNA, ss or ds
- calf spleen phosphodiesterase : cleaves off Np : Up > Gp = Ap >> Cp
- exonucleolytically from a 5'-OH of DNA er RNA, particularly on ss substrate
inhibited by secondary structure in ss RNA or DNA
- DNaseI (bovine pancreas)
=> nicking (dsDNA)
- nicking is quite random : produces a 5'-phosphate + 3'-OH
- Mg2+ required for nicking ; with Mn2+ instead of Mg2+ => second cleavage in
the opposing position of the complementary strand = ds cleavage
- with cccDNA : nicking is only once (=> ocDNA) in the presence of ethidium+
- application : 'nick translation' : labelling and tagging
- Nuclease S1 & Mung bean nuclease => cleave single strands endonucleolytically
- active on both DNA and RNA (if ss)
- optimal activity around pH 4 to 5 : yields 5'-phophate + 3'-OH
- Zn2+ required
- 'breathing' of the ends may lead to some extra shortening of the ds terminal parts ;
=> more difficult to control with nuclease S1 than with mung bean nuclease
- excise loop structures (confirming the endonucleolytical mechanism)
- ribonucleases : (crystallized ; very pure ; endoribonucleases)
- base-specific cleavages by RNase T1 (following Gp), RNase A (Yp),
RNase U2 (Rp), RNase T2 (Np) => (oligo)nucleotides with a
3'-terminal phosphate)
- hydrolysis via a 2'-3' cyclic intermediate (opening is the rate-limiting step)
- ring opening only by cleavage of the 2' linkage => only 3'-P oligonucleotides
- cleaving preferentially in single-stranded regions : hence partial digestions do not
generate genuinely random fragments, but more (secondary) structure-related overlaps.
- RNase T2 : some preference for adenylate linkages, but full cleavage produces
3'-mononucleotides (Np)
- RNase H (hybridase) : breaks the RNA strand in DNA:RNA heteroduplexes
wide occurrence in nature
- enzymes from bacteria and eukaryotic cells are endoribonucleases
- viral enzymes are (also?) exonucleases that stepwise remove 5'-mononucleotides
from both the 5' and 3' end
- a bond between a ribo- and a deoxyribonucleotide is not cleaved by the
bacterial RNaseH, but is cleaved by the viral and eukaryotic enzymes.